a) Field of the Invention
The invention is directed to a method in microscopy, particularly fluorescence microscopy, laser scanning microscopy, fluorescence correlation spectroscopy, and nearfield scanning microscopy, for the examination of predominantly biological specimens, preparations and associated components. This includes methods for screening active ingredients (high throughput screening) based on fluorescence detection. Simultaneous examinations of specimens with multiple fluorophores in real time by means of simultaneous illumination and/or detection of the specimen from both sides is possible.
b) Description of the Related Art
A typical area of application of light microscopy for examining biological preparations is fluorescence microscopy (Pawley, “Handbook of Biological Confocal Microscopy”, Plenum Press 1995). In this case, certain dyes are used for specific labeling of cell parts. The excitation of dyes is usually carried out by means of absorption of a high-energy photon (single-photon excitation). In addition to the excitation of dye molecules with a high-energy photon, excitation with a plurality of low-energy photons is also possible. This type of excitation of the dye is referred to as multiphoton absorption (Corle, Kino, “Confocal Scanning, Optical Microscopy and Related Imaging Systems”, Academic Press 1996).
A laser scanning microscope such as that described in DE19702753A1 is particularly suitable for the examination of thick preparations. From a three-dimensionally illuminated image, only the plane (optical section) located in the focal plane of the objective is reproduced by special detection arrangements in connection with the corresponding single-photon absorption or multiphoton absorption. By recording a plurality of optical sections in the x-y plane at different depths z of the specimen, a three-dimensional image of the specimen can be generated subsequently in computer-assisted manner.
Line scanners, as they are called, are also known from the prior art (Corle, Kino, “Confocal Scanning Optical Microscopy and Related Imaging Systems”, Academic Press 1996). The basic construction essentially corresponds to that of an LSM. However, instead of a point focus, a line is imaged in the specimen and the specimen to be examined is scanned in only one direction. The image acquisition rate can be substantially increased by scanning a line instead of a point. Therefore, this scanning method can be used for observing high-speed processes in real time (real time microscopy). Additional methods and arrangements for line scanners are described in DE 7505.
In another arrangement for real time microscopy according to the prior art, the entire field to be examined is illuminated by an expanded light source. However, only special point patterns of the total field to be scanned are uncovered by a rapidly rotating disk. These methods are usually referred to in technical literature as Nipkow disk methods (Corle, Kino, “Confocal Scanning Optical Microscopy and Related Imaging Systems”, Academic Press 1996).
In another method according to the prior art, known as structured illumination, the modulation depth of the optical imaging of an amplitude structure (e.g., grating) is used as a criterion for depth of field. For a detailed description reference is had to T. Wilson, et al., “Method of obtaining optical sectioning by using structured light in a conventional microscope”, Optics Letters 22 (24), 1997.
Different arrangements with double-objectives are known from the prior art. These arrangements make possible: 1) efficient collection of the dye fluorescence (see DE 19942998); 2) increased optical resolution with punctiform specimen illumination in a laser scanning microscope—4-Pi microscope (Schrader et al., Biophysical Journal , vol. 75, Oct. 1998, 1659-1668); 3) increased optical resolution in a widefield microscope with coherent specimen illumination, so-called standing wave microscope (F. Lanni, Applications of Fluorescence in the Biomedical Sciences, 1st ed., Liss, N.Y., 1986); and 4) increased optical resolution in a widefield microscope with incoherent specimen illumination, so-called I2M, I3M and I5M (M. G. L. Gustafsson, D. A. Agard ad J. W. Sedat, “I5M: 3D widefield light microscope with better than 100 nm axial resolution,” J. Microsc. (Oxford) 195, 10-16 (1999)).
The methods according to the prior art are disadvantageous in that the 4-Pi microscope, as point-scanning method, is slow and lateral structuring and therefore increased lateral resolution are impossible. The widefield methods require an incoherent light source for uniquely defined correlation of the object information. Further, the use of spatial filtering (“confocality”) is not possible, so that there is a high signal background in thick specimens resulting in a reduced signal-to-noise ratio. Further, high peak intensities are difficult to achieve for nonlinear specimen interaction without bleaching due to the small illumination surface.
In double-objective arrangements, the separation of the excitation light from light emitted by the specimen is carried out, according to the prior art, by spectral separation using Stokes shift, by limiting the numerical aperture of the optics used for specimen illumination or specimen detection, and by dividing into different polarization directions. For details on the prior art, reference is had to Pawley, “Handbook of Biological Confocal Microscopy” (Plenum Press 1995). The disadvantage in all of the methods according to the prior art, particularly when using double-objective arrangements, is that the separation of the excitation light from the light emitted by the specimen in double-objective arrangements is wavelength-dependent or is carried out with a limited efficiency of typically 70% to 90% depending on the required spectral characteristics and the quantity of illumination lines. When different wavelengths are to be used for exciting the dye fluorescences, the filters must be correspondingly adapted or changed, so that it is necessary to readjust the optical arrangements especially in case of interferometric superposition of the beams in a double-objective arrangement. In addition, the methods according to the prior art are not suitable for use in optical systems in which the beams impinge at a large inclination on the optical elements for separating because the spectral characteristics change, e.g., in a dichroic beam splitter, or worsen the efficiency of the polarizing division with a polarization splitter.